Abstract

TRAIL is an attractive anti-neoplastic agent due to its minimal toxicity and its potent ability to induce apoptosis in cancer cells. Clinical trials with intravenous administration of recombinant human TRAIL in cancer patients have been limited by the short serum half-life of TRAIL and molecular resistance mechanisms including loss of cell surface death receptor expression and/or pro-survival expression changes in proteins that regulate apoptosis such as Mcl-1. Although TRAIL death receptor-targeted agonistic antibodies have an increased half-life, they act on one of the two pro-apoptotic TRAIL receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In order to pharmacologically increase tumor and host TRAIL production as a novel anti-neoplastic mechanism, to improve the pharmacokinetic properties of TRAIL by increasing its effective half-life, and perhaps its local concentration within the tumor microenvironment, we screened for small molecules capable of inducing the endogenous TRAIL gene. A cell-based bioluminescence reporter screen was conducted in TRAIL-resistant Bax-null human HCT116 colon cancer cells using the TRAIL gene promoter yielded TRAIL-inducing compound 10 (TIC10). TIC10 induces TRAIL at the message, membrane-bound, and soluble levels in a time- and dose-dependent manner in TRAIL-sensitive and TRAIL-resistant human cancer cell lines as well as normal human fibroblasts without any effect on cell cycle profile. TIC10 induces cell death in a number of cancer cell lines without evidence of cell cycle arrest, a characteristic of HDAC inhibitors that are the only other pharmacological agent currently shown to upregulate TRAIL. Lack of structural homology with HDAC inhibitors along with this observation suggest a novel mechanism of TRAIL-upregulation. Western blot analysis revealed that TIC10 sensitizes to TRAIL-mediated apoptosis by tumor cell-specific upregulation of DR5 and downregulation of Mcl-1. Human xenograft experiments demonstrate that TIC10 is highly effective as a multi- or single-dose agent with similar efficacy to TRAIL in TRAIL-sensitive xenografts. Some TRAIL-resistant xenografts such as RKO demonstrate TIC10 superiority to TRAIL with no incidence of toxicity. TIC10 causes highly increased protein levels of TRAIL and cleaved caspase-8, increased TUNEL staining indicating apoptosis, in addition to striking decrease in tumor cell density by histology in tumors of human xenografts in mice. Mice lacking tumors demonstrate an upregulation of TRAIL protein levels in serum. The effect of TIC10 on normal cell production of TRAIL provides a novel mechanism for killing tumor cells through a bystander effect in the tumor microenvironment.